Human umbilical cord blood (UCB) can be a source of hematopoietic stem cells for gene therapy, as an alternative to allogenic bone-marrow transplantation, for the treatment of a number of genetic diseases.
Previous work has examined conditions for optimal retroviral mediated gene transduction of human bone marrow progenitor cells. It has been found that pre-stimulation of marrow (either the total mononuclear cell fraction or isolated CD34+ cells) with combinations of hematopoietic growth factors increases the extent of gene transfer. For example, IL-3 and IL-6 increased gene transfer from 10% to 50%. (Nolta, et al., Human Gene Therapy, Vol. 1, pgs. 257–268 (1990)). Addition of c-kit ligand provided a further increase in gene transfer efficiency. (Nolta, et al., Exp. Hematol., Vol. 20, pgs. 1065–1071 (1992)). The cytokines may act, at least in part, by inducing cell cycling which is required for retroviral integration. (Nolta, et al., 1990.)
The above studies were performed using the method of co-cultivation, in which the bone marrow cells are grown upon a monolayer of vector-producing fibroblasts. Initial attempts to obtain efficient gene transfer into human marrow progenitor cells using cell-free retroviral supernatants consistently yielded lower levels of gene transfer than can be achieved by using co-cultivation. Moore, et al., Blood, Vol. 79, pgs. 1393–1399 (1992) reported that the presence of an underlayer of marrow stromal cells facilitates efficient cell-free retroviral transduction of human marrow progenitor cells, which eliminated the need for co-culture on the murine vector packaging cells.
Umbilical cord and placental blood represent a uniquely rich source of hematopoietic stem cells. (Broxmeyer, et al., Proc. Nat. Acad. Sci., Vol. 86, pg. 3828 (1989)). Large numbers of stem cells can be collected from normal cord and placental blood without cytokine mobilization and without performing invasive procedures on the patient. Based on quantitative progenitor assays it has been estimated that there are a sufficient number of progenitors in a single cord/placental blood collection to reconstitute an adult after marrow ablation. (Broxmeyer, et al., Proc. Nat. Acad. Sci., Vol. 89, pg. 4109 (1992)). Within two days of birth, the number of circulating hematopoietic progenitor cells drops dramatically to the level seen in older children and adults; thus, collection of stem cells at birth from the cord or placenta represents a unique opportunity to obtain cells needed for gene therapy (such as, for example, gene therapy for ADA deficiency) easily and safely. Successful reconstitution of more than 50 children with hemoglobinopathies and with malignancies by allogenic cord blood has been performed. (Broxmeyer, et al., Blood Cells, Vol. 17, pg. 313 (1991); McGlave, Blood Cells, Vol. 17, pg. 330 (1991); Gluckman, et al., N. Engl. J. Med., Vol. 321, pg. 1174 (1989); Flomenberg, et al., Bone Marrow Transplant, Vol. 10, Suppl. 1, pg. 115 (1992)).
Some conditions which permit in vitro gene transfer into umbilical cord blood cells recently have been described. Moritz, et al., J. Exp. Med., Vol. 178, pgs. 529–536 (August 1993) found that co-cultivation of the cells was most efficient, viral supernatant transduction on a marrow stromal layer was almost as effective, and transduction with viral supernatant alone was less effective. These experiments also involved the use of c-kit ligand and Interleukin-6 in the culture medium.